We determined the influence of melatonin (MLT) on the induction of interleukin (IL)-2, IL-1β, IL-4, tumour necrosis factor-alpha (TNF-α) and gamma interferon (IFN-γ) on mice infected with the Venezuelan equine encephalomyelitis (VEE) virus. Levels of IFN-γ in the MLT-treated group were significantly increased ( P < 0·001) when compared with the control non-infected group from day 1 to 6 post-infection (p.i.), while in infected mice treated with 500 μg MLT/kg of bodyweight enhanced levels of IFN-γ were evident from 4 to 6 days p.i. No differences were detected in the levels of IL-2 between the controls, the infected mice treated with MLT and the infected untreated group, from day 2 p.i. No changes in serum levels of IL-4 were detected. In infected mice treated with MLT, blood levels of IL-1β were elevated almost 10-fold from day 1 to day 6 p.i. when compared to the control, the infected and the non-infected MLT-treated mice ( P < 0·001). A highly significant rise ( P < 0·001) of TNF-α was found in infected mice treated with MLT, from day 1 to 6 p.i. when compared to the other groups. A significant rise ( P < 0·001) was also evident in the infected non-MLT-treated group and a less pronounced rise in the non-infected mice treated with MLT when compared to controls. The blockade of IFN-γ and TNF-a did not inhibit the protective effect of MLT but when IL-1β was neutralized, 100% of the infected mice died suggesting that IL-1β induced by MLT treatment is a target cytokine to generate an immune response against the viral infection.